Patent Application
20220160716
The present invention relates to purine derivative compounds, in particular roscovitine analogues. The present invention also relates to roscovitine analogues according to the invention for use as medicaments. Finally, the present invention relates to roscovitine analogues for their use for treating rare biliary diseases like intrahepatic cholestatic diseases, and more particularly ABCB4-related biliary diseases, characterized by a defect of phosphatidylcholine secretion in hepatocytes.
Rare biliary diseases like intrahepatic cholestatic diseases are hereditary pathologies that both affect children's and adult's liver performance by inducing progressive liver disease, which typically leads to liver failure. Indeed, liver cells are less able to secrete the bile thus decreasing intrahepatic bile flow, which favours the formation of a toxic bile and leads to liver failure in affected individuals. Generally, hepatic cell destruction may also be a consequence of intrahepatic biliary obstruction.
These dysfunctions affecting bile formation may be due to deficiency and abnormal functioning of membrane transporters. Among them, Adenosine triphosphate Binding Cassette transporter, subfamily B member 4 (ABCB4), also called MDR3, plays a great role in liver performance. This member of the ATP Binding Cassette (ABC) superfamily is mainly expressed at the canalicular membrane of hepatocytes where its function is to translocate phosphatidylcholine (PC) from the inner leaflet to the outer leaflet of the hepatocyte canalicular plasma membrane, thus allowing PC secretion into bile. At the molecular level, secreted PC has an essential role since it forms mixed micelles with the other co-secreted hydrophobic bile components, such as bile salts and cholesterol. Indeed, impairment of PC secretion into bile leads to the formation of cholesterol crystals and gallstones, as well as loss of protection from the detergent effects of free bile salts on biological membranes of the biliary tree.
These clinical characteristics have been largely reported for patients with genetic variations of the ABCB4 gene (missense variations) which lead to rare biliary diseases, such as progressive familial intrahepatic cholestasis type 3 (PFIC3), low-phospholipid associated cholelithiasis (LPAC) syndrome or intrahepatic cholestasis of pregnancy (ICP). In the case of ABCB4-related biliary diseases, one of the reasons resulting in the impairment of PC secretion by ABCB4 transporter is the lack of cellular traffic preventing both its maturation and its plasma membrane localization. Therefore, this will induce both a retention of immature transporter in cell organelles responsible for protein's maturation and a deficiency in PC secretion.
For patients with ABCB4-related biliary diseases, the only pharmacological treatment is the administration of ursodeoxycholic acid (UDCA), a bile acid with low hydrophobicity. Although this therapy is efficient in the milder forms of the diseases, UDCA is not—or poorly—efficient in the majority of homozygous or compound heterozygous patients with severe forms of ABCB4-related diseases for whom the only alternative remains liver transplantation. In the absence of surgery and efficient pharmacological treatment able to cure patients, or at least delay liver transplantation, the life expectancy is low. Thus, pharmacological alternatives are eagerly needed.
One of the first alternatives considered is (R)-roscovitine, also known as Seliciclib or CYC202 and hereafter referred to as roscovitine. This 2,6,9-trisubstituted purine was identified as a relatively potent and selective Cyclin-Dependent Kinase (CDK) inhibitor. Roscovitine has undergone several studies in many indications up to clinical phase trials in various cancers, rheumatoid arthritis, glaucoma and cystic fibrosis. Interestingly, roscovitine has been shown to correct the intracellular localisation and the channel activity of CFTR, another ABC carrier family member involved in cystic fibrosis (EP2907514). Thus, roscovitine appeared as potential interesting treatment for ABCB4-related diseases. Moreover, studies show that treatment with 100 μM of roscovitine lead to increase maturation and relocation of three an ER-retained ABCB4 variants (ABCB4-I541F/I490T/L556R); retained in the endoplasmic reticulum-ER as an immature and high-mannose glycosylated protein.
However, roscovitine displayed important dose-dependent cytotoxicity, which might be explained by its CDK inhibition activity, and inhibition of ABCB4-WT-mediated phosphatidylcholine secretion activity in HEK cells.
Therefore, there is still a need to develop compounds that shows the same activity that roscovitine but without cytotoxicity for the treatment of intrahepatic cholestatic diseases such as PFICs.
In order to overcome cytotoxicity, the Applicant develop an alternative strategy by synthesizing less toxic roscovitine structural analogues. These compounds were able to correct the intracellular traffic, thereby restoring the maturation, the canalicular expression and more importantly the function of ABCB4 variants (I541F/I490T/L556R).
The invention thus relates to Compound of formula (I):
According to one embodiment, R1 is a (C1-C4) alkyl group selected from methyl, ethyl, propyl, isopropyl. According to one embodiment, R1 is isopropyl.
According to one embodiment, R1 is cyclopentyl.
According to one embodiment, R2 is a phenyl or substituted phenyl with one or two groups, said groups being selected from electron withdrawing groups and halogens.
According to one embodiment, R2 is a phenyl or a substituted phenyl with one or two groups, said groups being selected from CF3, F, Cl and Br.
According to this embodiment, R2 is a substituted phenyl with one to three groups and at least one group being in meta position. According to this embodiment, R2 is a substituted phenyl with one to two groups and at least one group being in meta position.
According to one embodiment, R1 is a cyclopentyl group and the group R2 is selected from:
According to one embodiment, the compound of formula (I) is selected from:
The invention further relates a composition comprising at least one compound of formula (I).
According to one embodiment, the composition is a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.
The invention further relates to the compound of formula (I) for use as a medicament.
The invention further relates to the composition according to the invention for use as a medicament.
According to one embodiment, the invention relates to the compound of formula (I) or the composition according to the invention for use in the treatment of rare biliary diseases such as intrahepatic cholestatic diseases.
According to another embodiment, the invention relates to the compound of formula (I) or the composition according to the invention for use in the treatment of intrahepatic cholestatic diseases being an ABCB4-related biliary disease, characterized by a defect of phosphatidylcholine secretion in hepatocytes.
According to another embodiment, the invention relates to the compound of formula (I) or the composition according to the invention for use in the treatment of progressive familial intrahepatic cholestasis (PFIC3), low phospholipid-associated cholelithiasis (LPAC) syndrome, intrahepatic cholestasis of pregnancy (ICP), drug-induced liver injury, transient neonatal cholestasis (TNC), adult biliary fibrosis and cirrhosis or intrahepatic cholangiocarcinoma (IHCC).
According to another embodiment, the invention relates to the compound of formula (I) or the composition according to the invention for use in the treatment of intrahepatic cholestatic diseases that are selected from the group consisting of: progressive familial intrahepatic cholestasis type 3, LPAC syndrome or ICP.
In the present invention, the following terms have the following meanings:
This invention relates to Compound of formula (I):
According to one embodiment, R1 is a (C1-C4) alkyl group. According to one embodiment, R1 is a (C1-C3) alkyl group. According to one embodiment, R1 is selected from methyl, ethyl, propyl and isopropyl. According to one embodiment, R1 is an isopropyl.
According to another embodiment, R1 is a (C3-C6) cycloalkyl group. According to one embodiment, R1 is a (C4-C6) cycloalkyl group. According to one embodiment, R1 is a (C5-C6) cycloalkyl group. According to one embodiment when R1 is selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexane. According to one embodiment, R1 is a cyclopentyl.
According to a one embodiment, R2 is a phenyl group.
According to another embodiment, R2 is a substituted phenyl with one to three groups. According to one embodiment, R2 is a substituted phenyl with one to three groups selected from electron withdrawing group and halogens. According to one embodiment, R2 is a substituted phenyl with one to three groups selected from CN, NO2, CF3, F, Cl, Br and I. According to one embodiment, R2 is a substituted phenyl with one to three groups selected from CF3, F, Cl and Br.
According to one embodiment, R2 is a substituted phenyl with one to two groups selected from electron withdrawing group and halogens. According to one embodiment, R2 is a substituted phenyl with one to two groups selected from CN, NO2, CF3, F, Cl, Br and I. According to one embodiment, R2 is a substituted phenyl with one to two groups selected from CF3, F, Cl and Br.
According to one embodiment, R2 is a substituted phenyl with one group selected from electron withdrawing group and halogens. According to one embodiment, R2 is a substituted phenyl with one group selected from CN, NO2, CF3, F, Cl, Br and I. According to one embodiment, R2 is a substituted phenyl with one group selected from CF3, F, Cl and Br.
According to this embodiment, when R2 is a substituted phenyl with one to two groups, at least one of said group is in meta position of the phenyl ring.
In one embodiment, R1 is a cyclopentyl group and the group R2 is selected from:
In one embodiment, R1 is an isopropyl and R2 is a 3-(trifluoromethyl)phenyl.
According to one embodiment, the compound of formula (I) is selected from:
According to one embodiment, the compound of formula (I) is selected from:
According to one embodiment, the compound of formula (I) is (R)-2-((9-cyclopentyl-6-((3-fluorobenzyl)amino)-9H-purin-2-yl)amino)butanoic acid:
According to a one embodiment, the compound of formula (I) is (R)-2-((6-((3-chlorobenzyl)amino)-9-cyclopentyl-9H-purin-2-yl)amino)butanoic acid:
According to a further embodiment, the compound of formula (I) is (R)-2-((6-((4-chloro-3-fluorobenzyl)amino)-9-cyclopentyl-9H-purin-2-yl)amino)butanoic acid:
The compounds were named using ChemBioDraw® Ultra version 12.0 (PerkinElmer).
The compounds of the invention may exist in the form of free bases or of addition salts with pharmaceutically acceptable acids. According to one embodiment, the compounds of the invention exist in the form of free bases. According to one embodiment, the compounds of the invention exist in the form of addition salts with pharmaceutically acceptable acids.
Suitable physiologically acceptable acid addition salts of compounds of formula (I) include hydrobromide, tartrate, citrate, trifluoroacetate, ascorbate, hydrochloride, triflate, maleate, mesylate, formate, acetate and fumarate.
The compounds of formula (I) and/or salts thereof may form solvates (e.g. hydrates) and the invention includes all such solvates.
The compound of the present invention can be prepared by conventional methods of organic chemistry practiced by those skilled in the art. In particular, the starting material may be 6-chloro-2-fluoropurine. One possible process is illustrated in example 1 hereinafter.
A further object of the invention is a medicament comprising, consisting or consisting essentially of at least one compound as described hereinabove.
Another object of the present invention is a composition comprising, consisting or consisting essentially of at least one compound as described hereinabove.
As used herein, “consisting essentially of”, with reference to a composition, means that at least one compound according to the invention, or combination thereof is the only one therapeutic agent or agent with a biologic activity within said composition.
Another object of the invention is a composition wherein said composition is a pharmaceutical composition comprising, consisting or consisting essentially of at least one compound as described hereinabove and at least one pharmaceutically acceptable excipient.
Examples of pharmaceutically acceptable excipients include, but are not limited to, media, solvents, coatings, isotonic and absorption delaying agents, additives, stabilizers, preservatives, surfactants, substances which inhibit enzymatic degradation, alcohols, pH controlling agents, and propellants.
Examples of pharmaceutically acceptable media include, but are not limited to, water, phosphate buffered saline, normal saline or other physiologically buffered saline, or other solvent such as glycol, glycerol, and oil such as olive oil or an injectable organic ester. A pharmaceutically acceptable medium can also contain liposomes or micelles, and can contain immunostimulating complexes prepared by mixing polypeptide or peptide antigens with detergent and a glycoside.
Examples of isotonic agents include, but are not limited to, sugars, sodium chloride, and the like. Examples of agents that delay absorption include, but are not limited to, aluminum monostearate and gelatin.
Examples of additives include, but are not limited to, mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration.
Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-Amine or NZ-Amine AS.
According to another aspect, the invention deals with the compound of formula (I) for use as a medicament.
According to another aspect, the invention deals with a composition comprising at least one compound of formula (I) for use as a medicament. In a more specific aspect, the composition is a pharmaceutical composition for use as a medicament comprising at least one pharmaceutically acceptable excipient.
The compound, the composition, the pharmaceutical composition or the medicament of the present invention may be administered orally, parenterally, by intraperitoneal administration, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
In one embodiment, the compound, the composition, the pharmaceutical composition or the medicament of the present invention is injected. Examples of injections include, but are not limited to, intratumoral, intradermal, subcutaneous, intravenous, intramuscular, intra-lymphatic, intra-articular, intra-synovial, intrasternal, intrathecal, intravesical, intravaginal, intrahepatic, intralesional and intracranial injection or infusion techniques.
In a further aspect of the invention, the compound or the composition is administrated via subcutaneous, intradermal, intraperitoneal or intravaginal routes.
In one embodiment, the compound, the composition, the pharmaceutical composition or the medicament of the present invention is in a form adapted to oral administration. According to a first embodiment, the form adapted to oral administration is a solid form selected from the group comprising tablets, pills, capsules, soft gelatin capsules, sugarcoated pills, orodispersing tablets, effervescent tablets or other solids. According to a second embodiment, the form adapted to oral administration is a liquid form, such as, for example, a drinkable solution, a buccal spray, liposomal forms and the like.
A compound of formula (I) according to the invention may be formulated with excipients and components that are commonly used for oral compositions, as for example, fatty and/or aqueous components, humectants, thickeners, preserving agents, texture agents, taste agents and/or coating agents, antioxidants, preserving agents.
The formulating agents and excipients for an oral composition are known in this field and will not be the object of a fully detailed description herein. Many embodiments of oral compositions are formulated via usual processes for producing coated tablets, gel capsules, gels, controlled-release hydrogels, emulsions, tablets and capsules.
Examples of coating materials include, but are not limited to, lecithin.
In another embodiment, strain, the compound, the pharmaceutical composition or the medicament of the present invention is formulated for rectal or vaginal administration and may be presented as suppositories, pessaries, tampons, creams, gels, pastes, foams or sprays.
An example of formulation of a rectal composition, is a suppository, containing conventional suppository bases, such as cocoa butter or other glycerides. In another embodiment, strain, the compound, the pharmaceutical composition or the medicament of this invention is in a form suitable for parenteral administration. Forms suitable for parenteral administrations include, but are not limited to, sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use.
Parenteral administration comprises subcutaneous, intra-muscular and intra-venous administration. Formulations for injection may be presented in single-unit dosage form, such as ampoules or in multi-dose containers. The compositions may be formulated as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain additional agents, such as preservatives, emulsifying and/or stabilizing agents. Alternatively, a compound of formula (I) may be formulated as a dispersible powder, which may be prepared as a liquid composition, with a suitable vehicle, for example sterile water, just before use.
In another embodiment, the compound, the pharmaceutical composition or the medicament of the invention is in a form adapted for local delivery via the nasal and respiratory routes. Examples of formulations suitable for nasal or respiratory administration include, but are not limited to, nasal solutions, sprays, aerosols and inhalants.
In another embodiment, the compound, the pharmaceutical composition or the medicament of the invention is in a form adapted to a topical administration. Examples of formulations adapted to a topical administration include, but are not limited to, ointment, paste, eye drops, cream, patch, such as, for example, transdermal patch, gel, liposomal forms and the like.
In one embodiment, the composition or formulation of the invention may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid excipient, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
A compound of formula (I) may be formulated as a liquid solution a liquid suspension or a powder.
As for liquid solution or suspension, a carrier may be typically pyrogen-free sterile or a dilute aqueous alcoholic solution. Liquid solution or suspension are preferably isotonic, hence may comprise sodium chloride. Optional additives include one or more preservative(s), such as for example methyl hydroxybenzoate, one or more antioxidant(s), one or more flavouring agent(s), one or more volatile oil(s), one or more buffering agent(s) and one or more surfactant(s).
As for a powder formulation, commonly used ingredients, such as a powdered diluent, for example powdered lactose, and surfactant(s) may be added.
Suitable formulations may also comprise one or more co-solvent(s), such as for example, ethanol, one or more surfactant(s), such as oleic acid and sorbitan trioleate, one or more antioxidant(s) and one or more suitable flavouring agent(s).
The exact dose for administration can be determined by the skilled practitioner, in light of factors related to the subject that requires treatment. Dosage is adjusted to provide sufficient levels of the composition or to maintain the desired effect of reducing signs or symptoms of the targeted pathologic condition or disorder, or reducing severity of the targeted pathologic condition or disorder. Factors which may be taken into account include the severity of the disease state (such as for example the tumor volume or the number of infected cells), the prognosis of the disease, the localization or accessibility to the tumor, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
In one embodiment, a therapeutically effective amount of the strain, the composition, the pharmaceutical composition, the medicament or the vaccine composition of the present invention is administered (or is to be administered) to the subject.
A dosage regimen suitable for the administration of a compound of formula (I) falls within the technical skills of an artisan in the art, and depends from multiple parameters. Indeed, a suitable dosage regimen depends from the gender, the age, the weight, and the progress of the disease. Within the scope of the instant invention, a suitable dosage regimen may encompass about 1 to 500 mg of the active compound.
For example, for an oral administration, a drug may comprise about 1 to about 500 mg of active compound, for example about 20 to about 250 mg of active compound, for example about 50 to 150 mg of active compound.
The present invention further relates to the compound, the composition, the pharmaceutical composition or the medicament, for use in preventing and/or treating severe biliary disease in a subject in need thereof. It also relates to methods of preventing and/or treating severe biliary diseases, by administering to a subject in need thereof the compound, the composition, the pharmaceutical composition, or the medicament according to the present invention.
In one embodiment, the strain, the composition, the pharmaceutical composition, the medicament or the vaccine composition is for treating severe biliary diseases.
In one embodiment, the compound, the composition, the pharmaceutical composition or the medicament is for use in treating and/or preventing intrahepatic cholestatic diseases.
In one embodiment, the compound, the composition, the pharmaceutical composition or the medicament is for use in treating and/or preventing an ABCB4-related biliary disease.
In a still further embodiment, the compound, the composition, the pharmaceutical composition or the medicament is for use in treating and/or preventing intrahepatic cholestatic diseases, progressive familial intrahepatic cholestasis (PFIC3), low phospholipid-associated cholelithiasis (LPAC) syndrome, intrahepatic cholestasis of pregnancy (ICP), drug-induced liver injury, transient neonatal cholestasis (TNC), adult biliary fibrosis and cirrhosis or intrahepatic cholangiocarcinoma (IHCC).
In a still further embodiment, the compound, the composition, the pharmaceutical composition or the medicament is for use in treating and/or preventing progressive familial intrahepatic cholestasis type 3, LPAC syndrome or ICP.
In one embodiment, the subject is a human.
In one embodiment, the subject has a severe biliary disease. In one embodiment, the subject is diagnosed or has been diagnosed with a severe biliary disease.
In another embodiment, the pharmaceutical composition according to the present invention may be administered prior, during or after another pharmaceutical composition comprising an additional agent such as, but not limited to, ursodeoxycholic acid (UDCA).
In a further embodiment, the pharmaceutical composition according to the present invention may comprise an additional agent.
In one embodiment, the subject was not treated previously with another treatment for a severe biliary disease (i.e., the method of the invention is the first line treatment).
In another embodiment, the subject previously received one, two or more other treatments for a severe biliary disease for example with UDCA (i.e., the method of the invention is a second line, a third line or more). In one embodiment, the subject previously received one or more other treatments for a severe biliary disease, but was unresponsive or did not respond adequately to these treatments, which means that there is no or too low therapeutic benefit induced by these treatments.
In another embodiment, the subject is at risk of developing a severe biliary disease. Examples of risk factors for developing a severe biliary disease include, but are not limited to, family history of a severe biliary disease or genetic predisposition.
The present invention is further illustrated by the following examples.
Compound 2 and compound 3 were prepared starting from 6-chloro-2-fluoropurine using a procedure starting from 2,6-dichloropurine (Oumata et al. 2009).
A mixture of compound 3 (1.7 mmol) and 3-aminobutanoic acid (12.26 mmol), K3PO4 (3.50 mmol) in 1 mL DMSO was heated at 160° C. during 5h. After cooling to 20° C. the mixture was diluted with 5 mL citric acid (10% in water, m:v). The mixture was extracted with EtOAc and the combined organic layers were washed with saturated NaCl and dried over Na2SO4. After evaporation of the solvent in vacuo, the crude product was purified on silica gel using CH2Cl2-EtOAc-THF (6:2:1).
The subcloning of wild type (WT) ABCB4, isoform A (NM_000443.3), into pcDNA3 vector has been described (6). The I541F, I490T and L556R missense ABCB4 variants were previously reported and described (2, 6, 11). Site directed mutagenesis were performed using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Les Ulis, France), following manufacturer's instructions, and using the primers (Eurogentec, Angers, France) described in Table 1 below. The sequences of all constructs were systematically verified by automated sequencing.
Human embryonic kidney (HEK-293, herein referred to as HEK; ATCC®-CRL-1573™) cells and human hepatocellular carcinoma HepG2 (ATCC®-HB-8065™) cells were obtained from ATCC (Manassas, Va.). As we previously reported, both HEK and HepG2 cells do not express ABCB4 (2, 12). Cells were grown in an incubator at 37° C. with 5% C02 in Dulbecco's Modified Eagle Medium (Gibco-Thermo Fisher Scientific, Villebon-sur-Yvette, France) containing 4.5 g/L D-glucose and supplemented with 10% heat-inactivated fetal bovine serum (Sigma, Saint-Quentin Fallavier, France), 2 mM L-glutamine, 2 mM sodium pyruvate, 100 units/mL of penicillin and 100 μg/mL streptomycin (Gibco-Thermo Fisher Scientific).
For transient transfection of HEK cells, they were seeded at subconfluent levels in the adequate tissue culture wells at least six hours before transfection. Turbofect (Thermo Fisher scientific) was used at a ratio of reagent:DNA of 2:1 according to manufacturer's instructions. For transient transfection of HepG2 cells, subconfluent cultures were seeded in the adequate culture wells 24 hours before transfection. Lipofectamine 3000 (Thermo Fisher Scientific) was used at a ratio of reagent:DNA of 1.5:1 according to manufacturer's instructions. Cell treatments and processing for further analyses were performed at least 16 hours post-transfection.
Cyclosporin A (CsA) was from Santa Cruz Biotechnologies (Dallas, Tex.). (R)-roscovitine and its non-commercial structural analogues (Aftin-4, the metabolite M3, MRT2-163, MRT2-164, MRT2-235, MRT2-237, MRT2-239, MRT2-241, MRT2-243, MRT2-245, MRT2-248 and MRT2-249; see
Protein kinases and their activators or regulators (see Table 2) were expressed and purified, and their catalytic activity was assayed in the presence of a range of concentrations of each roscovitine analogue, as described previously (8, 9, 15). IC50 values were calculated from the dose-response curves.
Western blots and indirect immunofluorescence were performed as previously described (2, 12). For immunoblot analyses, total cell lysates were prepared in denaturing and reducing sample buffer (13), separated on 7.5% SDS-PAGE and transferred on nitrocellulose membranes using Trans-Blot system (Bio-Rad Laboratories, Hercules, Calif.). Saturated membranes were incubated with mouse monoclonal anti-ABCB4 (clone P3II-26; Enzo Life Sciences, Villeurbanne, France) or anti-α-tubulin (clone 1E4C11; ProteinTech, Manchester, United Kingdom) antibodies and then with peroxidase conjugated anti-mouse secondary antibodies (Sigma). Signals were detected with ECL prime western blotting detection reagent (GE Healthcare, Velizy-Villacoublay, France) and quantified in the linear range of detection using ImageJ 1.50i software (U.S. National Institutes of Health, Bethesda, Md.).
For indirect immunofluorescence, HepG2 cells were grown on glass coverslips and after transfection and treatments, they were fixed and permeabilized during 1 min at −20° C. in ice-cold methanol. Then ABCB4 and ABCC2 were immunolabelled using the mouse monoclonal P3II-26 (IgG2b) and M2I-4 (IgG1) antibodies (Enzo Life Sciences), respectively. AlexaFluor® 555 and AlexaFluor® 488 conjugated isotype-specific secondary antibodies (Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific) were used to label ABCB4, ABCC2 and nuclei, respectively. Images were acquired using an IX83 inverted fluorescence microscope from Olympus (Rungis, France), equipped with a UPLSAPO 60XS2 silicone immersion objective and a Hamamatsu ORCA Flash4.0 digital CMOS camera, and analysed using Olympus CellSens Dimension Desktop version 1.16 and Adobe Photoshop version 8.0.1. For each experiment, all images were acquired with constant settings (acquisition time and correction of signal intensities).
Cell viability was assessed by the conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) into formazan crystals by living cells, as described (14). In brief, HEK cells were seeded in 96-well plates in triplicate for each tested condition, including controls (no cells, no treatment, treatment with vehicle). After transient expression of ABCB4-WT as described above (to mimic the experimental conditions of the other experiments) and drug treatment during 72 hours, 125 μg/ml MTT (final concentration) was added in each well and cells were re-incubated at 37° C. during 2 hours. These conditions were optimized in order to maintain the absorbance at 540 nm of the blanks below 0.1 OD unit (negative controls) and the absorbance at 540 nm for untreated cells between 1.0 and 1.3 OD units (positive controls). Then culture media were gently washed out, cells were lysed in 100 μL of pure DMSO and absorbance at 540 nm was measured using a multiplate cytofluorimeter SpectraFluor from Tecan (Männedorf, Switzerland). Cell viability was calculated for each triplicate after background subtraction and expressed as percentage of the mean for cells treated with vehicle only.
HEK cells were seeded in 0.01% poly-L-lysine (Sigma) pre-coated 6-well plates of 10 cm2 area per well (1.3×106 cells per well). After transient transfection of the empty vector (control), plasmids encoding ABCB4-WT or its variants, cells were incubated during 24 hours at 37° C. in serum-free medium supplemented with 0.5 mM sodium taurocholate (Sigma) and 0.02% fatty acid-free bovine serum albumin (Sigma). Then, the secreted PC was quantified from the collected media using a fluoro-enzymatic assay previously described (12). Each condition was analysed in triplicate, after background subtraction, and results were normalized to the expression levels of ABCB4 determined by immunoblots of the corresponding cell lysates.
Graphics and non-parametric analyses of variance tests (Kruskall-Wallis) tests were performed using Prism version 7.00 (GraphPad Software, La Jolla, Calif.). A P value of less than 0.05 was considered significant. If not specified in figure legends, symbols indicate the comparison between the control (or vehicle-treated) and the other tested conditions.
In this study, we used ABCB4-I541F, a prototypical ER-retained variant of ABCB4, first identified in a homozygous PFIC3 patient (1) and further characterized in our laboratory (3, 6). Indeed, ABCB4-I541F has been shown to be retained in the ER as an immature and high-mannose glycosylated protein (6), characterized by the absence or low abundance of a mature protein band on immunoblot, compared to the WT protein (
The cytotoxic effect induced by roscovitine might be explained by its CDK inhibition activity (16). We thus synthesized structural analogues (
1The activators/regulators of CDKs are indicated after the slashes: A, cyclin A; p25; T, cyclin T.
Interestingly, in HEK cells, some of these analogues (MRT2-235, -237, -239, -241, -243, -245 and -249) were able to significantly increase the expression of a mature form of ABCB4-I541F while other analogues (Aftin-4, M3, MRT2-163 and MRT2-164) were not (
To pursue this study, we decided to focus on three roscovitine analogues (MRT2-235, MRT2-237 and MRT2-243) which displayed significant rescue of the maturation and partial relocalisation of ABCB4-I541F at bile canaliculi (
It is also important to notice that treatment with roscovitine and these three analogues at 10, 25 and 100 μM do not significantly alter the plasma membrane localization of ABCB4-WT in HEK cells (
The inhibition of ABCB4 activity induced by MRT2-235, MRT2-237 and MRT2-243 when used at 100 μM led us to investigate these analogues at lower concentrations. After treatment with 5, 10 and 25 μM of the three analogues, we observed a dose-dependent correction of ABCB4-I541F maturation in HEK cells (
To extend this study, we analysed the effect of roscovitine analogues on two other ER-retained missense ABCB4 variants, I490T and L556R, identified in patients with liver cancer (10) or PFIC3(1). Without treatment, these variants were mainly detected as immature proteins by immunoblot in HEK cells (
| Number | Date | Country | Kind |
|---|---|---|---|
| 19305340.2 | Mar 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/057833 | 3/20/2020 | WO | 00 |
